Short‐chain fatty acids profile in patients with SARS‐CoV‐2: A case‐control study

Abstract Background and Aims SARS‐CoV‐2, as a new pandemic disease, affected the world. Short‐chain fatty acids (SCFAs) such as acetic, propionic, and butyric acids are the main metabolites of human gut microbiota. The positive effects of SCFAs have been shown in infections caused by respiratory syncytial virus, adenovirus, influenza, and rhinovirus. Therefore, this study aimed to evaluate the concentration of SCFAs in patients with SARS‐CoV‐2 compared with the healthy group. Methods This research was designed based on a case and control study. Twenty healthy individuals as the control group and 20 persons admitted to the hospital with a positive test of coronavirus disease (COVID‐19) real‐time polymerase chain reaction were included in the study as the patient group from September 2021 to October 2021, in Tabriz, Iran. Stool specimens were collected from volunteers, and analysis of SCFAs was carried out by a high‐performance liquid chromatography system. Results The amount of acetic acid in the healthy group was 67.88 ± 23.09 μmol/g, while in the group of patients with COVID‐19 was 37.04 ± 13.29 μmol/g. Therefore, the concentration of acetic acid in the patient group was significantly (p < 0.001) lower than in the healthy group. Propionic and butyric acid were present in a higher amount in the control group compared with the case group; however, this value was not statistically significant (p > 0.05). Conclusion This study showed that the concentration of acetic acid as the metabolite caused by gut microbiota is significantly disturbed in patients with COVID‐19. Therefore, therapeutic interventions based on gut microbiota metabolites in future research may be effective against COVID‐19.

higher concentrations than propionate and butyrate in the intestine, and the concentration of propionate and butyrate are almost equal. 7 The impact of SCFAs compounds in the intestine are including resource of energy production for colonocytes, anti-inflammatory role, and regulation of immune system responses. 8 The remarkable point is that the effects of these compounds are not only limited to the intestines and digestive system. From this perspective, SCFAs can affect other parts of the body, such as the lungs by entering the bloodstream. 9 Several studies have shown the protective effect of SCFAs against infectious agents. The mechanism of action can be directly on the infectious agent or indirectly through the immune system. 10,11 In previous studies, the positive effects of SCFAs have been shown for infections caused by respiratory syncytial virus (RSV), adenovirus, influenza, and rhinovirus. 12,13 Meanwhile, the relationship between SCFAs and COVID-19 disease is less understood.
Therefore, this study aimed to evaluate the concentration of SCFAs in patients with SARS-CoV-2 compared with the healthy group.

| Study design and sample selection
This research was designed based on a case and control study. For this purpose, 20 healthy individuals as the control group, and 20 persons admitted to the hospital with a positive real-time polymerase chain reaction test of COVID-19 were included in the study as a case group from September 2021 to October 2021, in Tabriz, Iran. After matching the age and gender, healthy volunteers were selected as the control group. The exclusion criteria for both groups were taking any antibiotics and probiotics for at least 2 months before sampling.
Stool samples were collected and immediately stored at −80°C until to do the test.

| Fecal SCFAs extraction
SCFA extraction and high-performance liquid chromatography (HPLC) analysis were performed based on a previous study with some changes. 14 At first, 300 mg stool sample was transferred to a 2 mL microtube and mixed with 1 mL of deionized distilled water, and centrifuged at 12,000 rpm for 10 min. Then, 100 μL of absolute HCl was added to the supernatant solution. Also, 5 mL of diethyl ether was added to this solution and placed on the rotator for 20 min, and centrifugation was performed for 5 min at 3500 rpm. After that, 500 μL of NaOH 1 N was added to the organic phase. The centrifugation was carried out for 5 min at 3500 rpm again, and 100 μL of absolute HCl was mixed with the sediment and was vortexed. Finally, after filtration, this solution was ready to be injected into the HPLC system.

| Statistical analysis
Data analyses were carried out using GraphPad software 9.4.1.
Descriptive statistics were conducted to assess the demographic and clinical variables. Analytical statistics were used to compare the mean concentration of SCFAs in two groups. For this purpose, the data were evaluated for normality using the Shapiro-Wilk and Kolmogorov-Smirnov tests. For normal data, the student's t test was used to compare the mean in two groups, and Mann-Whitney U test was used for nonnormal data. Quantitative values were reported based on the mean ± standard deviation, and a p < 0.05% was considered for significant results. Table 1 shows some demographic information of the patient group and healthy group. As shown, there is no significant difference concerning age and gender variables in the two groups. The mean age of the patient group was 55.40 ± 13.21 years, and the healthy group was 54 ± 12.55 years old.

| RESULTS
In general, in both groups, the concentration of acetic acid was higher than propionic and butyric acid. The amount of acetic acid in the healthy group was 67.88 ± 23.09 μmol/g, while in the group of patients with COVID-19, this value was 37.04 ± 13.29 μmol/g. Therefore, the concentration of acetic acid in the patient group was significantly lower than in the healthy group (p < 0.001, Figure 1A). Propionic acid was observed in the healthy group with a concentration of 1.56 ± 0.26 μmol/g, and in the patient group, this amount was 1.43 ± 0.35 μmol/g. The proportion of propionic acid in the healthy group was higher than that in the patient group, but this value was not statistically significant (p > 0.05, Figure 1B). The amount of butyric acid in the healthy group was 1.54 ± 0.30 μmol/g, and that in the patients with COVID-19 was 1.34 ± 0.32 μmol/g. Similar to propionic acid, the concentration of butyric acid was higher in the healthy group, but there was not a statistically significant relationship (p > 0.05, Figure 1C).

| DISCUSSION
In recent years, it became clear that the impact of gut microbiota on human health is undeniable. 15  Our results showed significant changes in the concentration of SCFAs between the two groups ( Figure 1). In both groups, the concentration of acetic acid was much higher than propionic and butyric acid. Acetic acid has two carbon atoms and is the shortest fatty acid in the intestine. The effects of energy production, maintaining body homeostasis, improving the immune system, and the anti-inflammatory role of acetic acid have been shown in various studies. 16 18 Second, these metabolites lead to signal transmission from the G protein-coupled receptors route. 19 This activity leads to the expression of GPR109A and GPR43 on macrophages, neutrophils, and dendritic cells, and the consequences of this action ultimately improve the function of immune system cells. 20 The main challenge in SARS-CoV-2 infections is the overexpression of ACE-2 receptors, which is influenced by factors such as diabetes, cardiovascular diseases, and smoking.
Evidence shows that propionate and butyrate lead to a decrease in the expression of ACE-2 receptor through the effect on cell signaling. 21,22 Beyond this effect, Takabayashi et al. showed that propionate and butyrate with other SCFAs even lead to a reduction in SARS-CoV-2 genome expression. 23 However, contrary to these results, our study showed that although the concentration of propionate and butyrate was higher in the healthy group, the difference was not statistically significant. Therefore, the findings related to propionate and butyrate in this study were not in agreement with the previous investigation. Among the main reasons for this difference, we can point to factors such as microbiota diversity, type of diet, and host genotype. 24

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
All volunteers who entered this study signed the informed consent

TRANSPARENCY STATEMENT
The lead author Reza Ghotaslou affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.